基于ITS基因分析黑斑蛙裂头蚴种系发育关系

Phylogenetic analysis of ribosomal ITS gene of frog Sparganum in Hunan province

为了研究黑斑蛙裂头蚴种系发育关系,本研究利用聚合酶链式反应(PCR)对黑斑蛙裂头蚴湖南株核糖体DNA内转录间隔区(ITS)进行扩增与测序,应用ClustalX 2.0程序对序列进行比对,再利用Mega4.0程序进行NJ法绘制种系发育树,研究黑斑蛙裂头蚴与其他带科绦虫的种群遗传关系。结果显示,湖南省不同地区10个黑斑蛙裂头蚴分离株ITS序列基本一致,为1 362~1 382bp,各分离株之间ITS-1和ITS-2序列的同源性均高于95.4%和94.6%;与基因库中其他带科绦虫ITS-1和ITS-2序列的同源性均低于59.5%和56.9%。通过建立NJ系统发育树,湖南省黑斑蛙裂头蚴分离株与欧猬迭宫绦虫位于同一大分支,得到了很好的鉴定。黑斑蛙ITS序列在种内相对保守,而种间差异较大,因此ITS序列可以作为分子标记研究黑斑蛙裂头蚴种系遗传变异,从而为黑斑蛙裂头蚴的分子流行病学和群体遗传研究奠定基础。

The objectives of the present study were to analyze sequence variation of the ribosomal ITS among frog sparganum isolates from Hunan province, The ITS was amplified from each spar- ganum isolates by PCR, and the ITS sequences were aligned using the ClustalX 2.0. The results showed that the lengths of ITS sequences were 1 362-1 382 bp. The similarities of the correspond- ing sequences were more than 95.4（ITS-1）and 94.6% （ITS-2）among isolates from Hunan prow ince,and the similarity of Hunan isolates and reference of other cestode strains in GenBank was lower than 59. 5% （ITS-l）and 56.9% （ITS-2）. The constructed phylogenetic tree revealed that the Hunan isolates and the Spirometraerinaceieuropaei available in GenBank were clustered in the same clade. It is concluded that ITS sequences can be used as a genetic marker for species genetic variation of frog sparganum. The results of the present study provided a useful basis for further studies of molecular epidemiology of frog sparganum,and diagnosis of the resultant disease.